Maintenance of a clear, healthy cornea is essential for vision. Persistent corneal wounds and pathogens that bring about corneal ulceration can and often do lead to blindness. Mesenchymal-epithelial interactions have been shown to occur in developing, healing, ulcerating and diseased corneas. However, there has been no systematic monitoring of the synthetic events or the endogenous factors that pass between tissue layers in these states. The objective of this proposal is to identify the molecular components that pass between the adult corneal stroma and corneal epithelium to modulate their interaction in normal, repair and disease states. Specifically, the aims of the projects are to determine the proteins and glycoproteins that are synthesized in the stroma and epithelium in response to trauma or disease and to identify the molecules that pass between tissue layers. These corneal stromal-epithelial interactions will be examined in normal corneas and in four stressed states. The stressed states will be: 1) Alteration of stromal cell synthesis: Protein synthesis of the keratocytes will be blocked by injection into the stroma of Muconomycin A, an irreversible protein synthesis inhibitor. 2) Removal of basement membrane: Keratectomies will be performed to allow direct contact of the epithelium and stromal matrix. 3) Sterile ulceration: Ulceration will be induced by thermal burn. 4) Corneal vascularization: Neovascularization will be induced by injection of urokinase. Changes in synthetic rates will be monitored by incorporation of [3H] leucine, [3H] glucosamine and [3H] proline. Newly synthesized molecules will be assayed using 2-D gel electrophoresis and fluorography. Molecules that pass between the stroma and epithelium will be detected by recombining denuded stroma and epithelial sheets in which only one tissue layer is radiolabeled. Radiolabeled molecules that pass into the opposite layer will be analyzed with HPLC and 2-D gels. Muconomycin A will be used to treat one layer, ensuring that any labeled molecules must have been transmitted from the other layer. By understanding whta newly biosynthesized molecules pass between tissue layers of the eye during disease and repair, one can potentially design better therapies that may intervene at an early stage of repair, making the processes occur more rapidly, and/or with less tissue damage.